Selpercatinib combination with the mitochondria-targeted antioxidant MitoQ effectively suppresses RET–mutant thyroid cancer

Genetic alternation of REarranged during Transfection (RET) that leads to constitutive RET activation is a crucial etiological factor for thyroid cancer. RET is known to regulate mitochondrial processes, although the underlying molecular mechanisms remain unclear. We previously showed that the multi-kinase inhibitors vandetanib and cabozantinib increase the mitochondrial membrane potential (Δψm) in RET-mutated thyroid tumor cells and that this effect can be exploited to increase mitochondrial enrichment of Δψm-sensitive agents in the tumor cells. In this study, we hypothesized that the RET-selective inhibitor, selpercatinib, can increase Δψm and, subsequently, tumor cell uptake of the mitochondria-targeted ubiquinone (MitoQ) to the level to break the mitochondrial homeostasis and induce lethal responses in RET-mutated thyroid tumor cells. We show that selpercatinib significantly increased Δψm, and its combination with MitoQ synergistically suppressed RET-mutated human thyroid tumor cells, which we validated using RET-targeted genetic approaches. Selpercatinib and MitoQ, in combination, also suppressed CCDC6-RET fusion cell line xenografts in mice and prolonged animal survival more effectively than single treatments of each agent. Moreover, we treated two patients with CCDC6-RET or RETM918T thyroid cancer, who could not take selpercatinib at regular doses due to adverse effects, with a dose-reduced selpercatinib and MitoQ combination. In response to this combination therapy, both patients showed tumor reduction. The quality of life of one patient significantly improved over a year until the tumor relapsed. This combination of selpercatinib with MitoQ may have therapeutic potential for patients with RET-mutated tumors and intolerant to regular selpercatinib doses.


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Wenjing Chen 1 , Sophie Dream 2 , Pui-Yin Leung 1 , Pui-Kei Wu 1 , Stuart Wong 3 & Jong-In Park 1 Genetic alternation of REarranged during Transfection (RET) that leads to constitutive RET activation is a crucial etiological factor for thyroid cancer.RET is known to regulate mitochondrial processes, although the underlying molecular mechanisms remain unclear.We previously showed that the multikinase inhibitors vandetanib and cabozantinib increase the mitochondrial membrane potential (Δψ m ) in RET-mutated thyroid tumor cells and that this effect can be exploited to increase mitochondrial enrichment of Δψ m -sensitive agents in the tumor cells.In this study, we hypothesized that the RETselective inhibitor, selpercatinib, can increase Δψ m and, subsequently, tumor cell uptake of the mitochondria-targeted ubiquinone (MitoQ) to the level to break the mitochondrial homeostasis and induce lethal responses in RET-mutated thyroid tumor cells.We show that selpercatinib significantly increased Δψ m , and its combination with MitoQ synergistically suppressed RET-mutated human thyroid tumor cells, which we validated using RET-targeted genetic approaches.Selpercatinib and MitoQ, in combination, also suppressed CCDC6-RET fusion cell line xenografts in mice and prolonged animal survival more effectively than single treatments of each agent.Moreover, we treated two patients with CCDC6-RET or RET M918T thyroid cancer, who could not take selpercatinib at regular doses due to adverse effects, with a dose-reduced selpercatinib and MitoQ combination.In response to this combination therapy, both patients showed tumor reduction.The quality of life of one patient significantly improved over a year until the tumor relapsed.This combination of selpercatinib with MitoQ may have therapeutic potential for patients with RET-mutated tumors and intolerant to regular selpercatinib doses.
Upon activation, RET promotes various cellular processes, including cell growth, proliferation, and survival, mainly through the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK), the phosphatidylinositol-3 kinase/protein kinase B (AKT) 16 , and the Janus kinase/signal transducer and activator of transcription pathways 2 .
RET mutation is an actionable therapeutic target.While vandetanib and cabozantinib have been approved by the Food and Drug Administration (FDA) for RET-mutant MTC and RET fusion-positive differentiated thyroid cancer [17][18][19] , these inhibitors are not RET-selective and ineffective against RET V804 gatekeeper mutations.Selpercatinib (RETEVMO™) and pralsetinib (GAVRETO™) are highly selective RET inhibitors designed to overcome the gatekeeper mutations 20,21 and approved by the FDA in 2020 for RET-mutant thyroid and lung cancers [22][23][24] .Moreover, in September 2022, the FDA granted accelerated approval to selpercatinib for tumoragnostic treatment of adult patients with locally advanced or metastatic solid RET tumors 25 .While generally very well tolerated, these inhibitors cause adverse effects in patients, including hypertension and hepatotoxicity as the most common adverse effects 23,24 .These adverse effects cause permanent discontinuation of selpercatinib in about 5%, dosage interruptions in about 42%, and dose reductions in about 31% of the patients 23 .Therefore, it is important to design a strategy to use the drug effectively while reducing its adverse effects.
We previously showed that low-dose vandetanib and cabozantinib can synergize with mitochondria-targeted agents to suppress MEN2A and MEN2B MTC cells 26 .Mechanistically, these inhibitors increased mitochondrial membrane potential (Δψ m ) in the RET tumor cells, subsequently facilitating tumor cell uptake and retention of Δψ m -sensitive triphenyl phosphonium (TPP)-conjugated carboxy-proxyl (MitoCP) and ubiquinone (MitoQ).Consequent enrichment of MitoCP and MitoQ beyond tumor cell tolerance disrupted mitochondrial homeostasis, triggering tumor cell death 26,27 .The TPP moiety has been used as a vehicle for mitochondria targeting of different functional moieties 28,29 .Of note, while MitoQ is currently used as a dietary supplement due to its beneficial effects on mitochondrial bioenergetics 30,31 and vascular function [32][33][34] , it also suppresses tumor cells derived from different tumors, including MTC 26 , melanoma 35 , breast cancer [36][37][38] , and pancreatic cancer 39 .This tumor suppressive effect is mainly attributed to higher MitoQ accumulation in tumor cells due to higher basal Δψ m in tumor cells.This information suggests that MitoQ could be a safe agent for tumor suppression if its tumor cell enrichment is selectively facilitated.
In this study, we reasoned that Δψ m -inducing effects of vandetanib and cabozantinib are due to RET inhibition and hypothesized that a low dose of selpercatinib will also increase Δψ m in RET-mutated tumor cells, priming the tumor cells to MitoQ sensitivity.We demonstrate that a combination of selpercatinib and MitoQ can effectively and synergistically suppress RETmutated thyroid cancers.

Results
Selpercatinib increases Δψ m in CCDC6-RET fusion mutated PTC cells We determined whether selpercatinib can increase Δψ m in TPC1 cells, the only human PTC cell line that carries a coiled-coil domain containing 6 (CCDC6)-RET fusion mutation, which is also known as RET-PTC1 40 .As determined by a dose-dependent analysis, we found that 72-hour treatment of selpercatinib between 1 nM and 1 µM dose ranges significantly decreased TPC1 cell viability with IC 50 of 15 nM (Supplementary Fig. 1a).Western blot analysis of these cells revealed that selpercatinib substantially decreased RET phosphorylation at Tyr905, an activating autophosphorylation 16,41,42 , and phosphorylation of ERK1/2 and AKT, the key downstream effectors of RET 16 , while increasing expression of the cyclin-dependent kinase inhibitor p27 KIP1 but decreasing p21 CIP1 expression (Supplementary Fig. 1b, c).These effects are consistent with those of other RET inhibitors in this cell line 43,44 .Under these conditions validating selpercatinib potency, TPC1 cells stained with tetramethyl-rhodamine methyl ester (TMRM), a Δψ m -sensitive fluorescent dye, significantly increased in a selpercatinib dose-dependent manner (Fig. 1a, b).We confirmed that these selpercatinib effects are specific to the depletion of RET activity using RNA interference.Similar as selpercatinib, two short hairpin RNA (shRNA) constructs targeting different regions of RET mRNA consistently increased Δψ m in TPC1 cells (Fig. 1c, d), while inducing growth inhibitory effects (Supplementary Fig. 1d-f).To further determine RET-specificity of this effect, we also used the immortalized normal rat thyrocytes PCCL3 expressing a doxycycline-inducible human nuclear receptor coactivator 4 (NCOA4)-RET 45 , which is the second most common RET fusion in PTC 46 .Indeed, selpercatinib increased Δψ m in this cell line model only upon doxycycline treatment (Fig. 1e, f).These data suggest that depletion of RET activity increases Δψ m in RET-mutated PTC cells.
Next, we determined whether selpercatinib can sufficiently increase mitochondrial enrichment of a Δψ m -sensitive agent, using MitoCP as a tool compound.MitoCP contains TPP and the CP moiety, a 5-membered nitroxide free radical that can form a covalent conjugate with a thiol protein, which can be detected by a TPP-specific antibody 26,47 .For this, TPC1 cells pretreated with selpercatinib, were treated with MitoCP for 1 hour.When mitochondria fractions extracted from selpercatinib-pretreated cells were compared with the fractions extracted from unpretreated cells, much higher levels of TPP-protein adducts were detected by Western blotting in the cells pretreated with selpercatinib (Fig. 1g, h).Consistently, RET knockdown also increased TPP-protein adducts in TPC1 cells (Fig. 1i, j).These data suggest that selpercatinib can increase mitochondrial enrichment of a Δψ m -sensitive agent in RET-mutated tumor cells, similar as vandetanib and cabozantinib 26 .

Selpercatinib and MitoQ can synergistically suppress the viability of RET-mutant PTC and MTC cells in culture
As the first step to evaluate the efficacy of MitoQ and its combination with selpercatinib in RET-mutant tumor cells, we validated that mitochondrial enrichment of its functional moiety, ubiquinone, is critical for its growth inhibitory effects in TPC1 cells.As determined by the crystal violet staining, MitoQ treatment for 48 hours significantly decreased TPC1 cell viability, exhibiting the IC 50 value at 250 nM (Fig. 2a).In contrast, the functional moiety, CoQ10, did not significantly affect the cell viability while the vehicle, TPP, decreased cell viability only mildly at higher doses when these compounds were compared with MitoQ in identical dose ranges (Fig. 2a).
Having established the necessity of mitochondrial delivery of CoQ10 for suppressing TPC1 cell viability, we determined whether selpercatinib can synergize with MitoQ to suppress TPC1 cell viability in vitro.As determined by crystal violet staining, the combination of 24-hour selpercatinib pretreatment with a subsequent MitoQ treatment induced a robust viability loss in TPC1 cells (Fig. 2b).Of note, our analysis using Synergy-Finder 2.0 48 suggested that this viability loss is attributed to a synergy between these two agents (Fig. 2c and Supplementary Fig. 2).Consistent with this effect, a combination of RET knockdown and MitoQ treatment also effectively suppressed TPC1 cell viability (Fig. 2d), with a combination index (CI) suggesting synergy (Fig. 2e), as determined by Compusyn 49 .Importantly, tumors in clinical contexts are generally under hypoxic conditions 50 .We therefore determined whether the selpercatinib-MitoQ combination produces similar synergistic effects in TPC1 cells in Human Plasma-Like Medium under a hypoxic culture condition, which was validated by increased HIF1α expression (Fig. 2f).Indeed, selpercatinib and MitoQ synergistically suppressed the viability of TPC1 cells under this hypoxic condition (Fig. 2g, h), which is consistent with their effects under the normoxic culture condition.
These combinatory effects were not limited to TPC1 cells.The combination of 24-hour selpercatinib pretreatment with the subsequent MitoQ treatment also synergistically suppressed the viability of the human MTC cell lines, TT (RET C634W ) and MZ-CRC-1 (RET M918T ), in cultures, as determined by crystal violet staining (Fig. 2i, j; Supplementary Fig. 2).Consistently, RET knockdown also increased Δψ m in these cells (Supplementary Fig. 3a, b).Moreover, overexpression of wild-type RET, RET E632D/L633V/C634R , and RET M918T rendered HEK293 cells to increase Δψ m upon selpercatinib treatment (Supplementary Fig. 3c, d), demonstrating that the selpercatinib on Δψ m effect can be reconstituted in normal cells by increasing RET activity.These data strongly suggest that depletion of RET activity can synergize with MitoQ to suppress RET-mutant tumor cells.

A combination of selpercatinib and MitoQ effectively suppresses the growth of TPC1 xenografts in mice
We determined the preclinical efficacy of the selpercatinib and MitoQ combination using immune-compromised mice bearing TPC1 xenografts.These animals were subjected to 4 cycles of treatments wherein selpercatinib and MitoQ were orally administered singly or in combination.For the drug combination, each cycle consisted of 2-day selpercatinib treatment followed by 2-day MitoQ treatment and 1 drug holiday (Fig. 3a), which is similar to the schedule that we previously used for preclinical evaluation of the vandetanib and MitoCP combination [23].For the comparison between monoand combination therapies, we used selpercatinib at 0.5 mg/kg/dose, a much lower dose than the highly potent doses (16-30 mg/kg twice a day) in different preclinical models 51,52 , because its standard doses were too potent to determine the effect of its combination with other drugs (Supplementary Fig. 4).Likewise, we used MitoQ at 20 mg/kg/dose, which did not significantly reduce tumor volume in a preclinical breast cancer model albeit decreasing tumor metastasis 37 .
Consistent with the in vitro data above, the sequential administration of selpercatinib and MitoQ most effectively suppressed the growth of TPC1 xenografts when compared with the monotherapy groups (Fig. 3b), and significantly prolonged mouse survival (Fig. 3c) without affecting body weight (Fig. 3d).In contrast, the selpercatinib monotherapy group exhibited partial tumor suppression while the MitoQ monotherapy group did not show any difference from the control group.Although we also analyzed tumors by Western blotting, we could not detect any significant changes in the cleavage of poly (ADP-ribose) polymerase (PARP) and lamin A, expression of p27 KIP1 , p21 CIP1 , and cytochrome c oxidase subunit 4 (COX IV), and the activation phosphorylation of RET, ERK1/2, and AKT (Fig. 3e), which are the surrogate markers for cell death, cell cycle arrest, mitochondrial integrity, and RET signaling activity, respectively.These data suggest that the combination effect may be independent of these proteins, although the different timing of tumor harvest complicates the interpretation of these data.
Patients with RET-mutant thyroid cancer tolerate and respond to combination therapy using selpercatinib and MitoQ We treated two patients with RET-mutant thyroid cancer with a selpercatinib and MitoQ combination.First patient was a 55-year-old man with poorly differentiated metastatic PTC with a history of poorly controlled hypertension.His tumor progressed after surgical removal of tumor mass and 153.8 mCi of I-131 for 4 weeks (Fig. 4a, full case report in the supplemental document).While awaiting approval for next-generation sequencing (NGS), the patient was treated with lenvatinib but this therapy was held after 5 weeks due to exacerbation of pre-existing refractory hypertension, grade 3, and symptoms of right heart failure (Fig. 4b).While on lenvatinib and during the hold, his cancer continued to progress and his heart dysfunction worsened (Fig. 4b, c).Because of the deteriorating medical status of this patient and the presence of CCDC6-RET fusion mutation in his tumor, we started a weekly cycled schedule for reduced selpercatinib dose with the addition of MitoQ: selpercatinib 160 mg twice a day (BID) on days 1, 2 (off days 3, 4, 5, 6, 7) followed by MitoQ 20 mg daily (QD) on days 3, 4, 5, 6, 7 (off days 1, 2) (Fig. 4a).The patient responded well to this treatment for 22 months, with a sustained radiographic partial response evaluated by response evaluation criteria in solid tumors (RECIST) following the first four months of treatment (Fig. 4c).A chest wall skin metastasis completely resolved during treatment (Fig. 4d).However, at 23 months of treatment, neck and chest CT scan revealed a new site of oligometastatic disease in the manubrium, which progressed over the next month.
The second patient was a 34-year-old female with RET M918T locally advanced, unresectable MTC, exhibiting elevated carcinoembryonic antigen and calcitonin levels.This patient was initially started on selpercatinib 160 mg BID for 7 weeks (Fig. 5a, full case report in the supplemental document) but this treatment was on hold due to hepatotoxicity with elevated asymptomatic alanine transferase (ALT) and aspartate transferase (AST) (Fig. 5b).Selpercatinib was resumed in accordance with the FDAapproved package insert to dose level -2, 80 mg BID after liver function tests normalized (Fig. 5a).However, she briskly developed recurrent grade 2 transaminase elevation, nearly grade 3, soon after resumption of treatment (Fig. 5b).Therapy would normally be permanently discontinued after a second occurrence of grade 3/4 hepatotoxicity.However, due to her critical situation, selpercatinib was resumed in a reduced dose schedule with the addition of MitoQ using the following weekly cycled schedule: selpercatinib 80 mg BID on days 1, 2 (off days 3-7) followed by MitoQ 10 mg QD on days 3-7 (off days 1 and 2) (Fig. 5a).Calcitonin levels decreased after full dosage of selpercatinib, elevated again at reduced dose of selpercatinib, and decreased and stabilized during the combination therapy (Fig. 5c).Followup imaging after 7 weeks on this regimen indicated further reduction in tumor size with achievement of partial response by RECIST criteria (Fig. 5d).The treatment continued with the plateau of response on imaging at 24 weeks of treatment.Her treatment response and reduction in tumor size allowed her to undergo curative intent surgical resection.

Discussion
Previous studies demonstrated that many tumor cells, mainly derived from the cancers of thyroid, skin, and breast, are more sensitive to MitoQ than normal cells 35,36,38 .MitoQ sensitivity of these tumor cells is mainly attributed to the elevated steady-state Δψ m of tumor cells, which is required to enhance their capacity to adapt to hypoxia, escape anoikis, and increase invasiveness 53,54 .Because the cationic TPP moiety of MitoQ is sensitive to Δψ m , TPP-conjugated agents would accumulate higher in the mitochondria of tumor cells than those of most normal cell types 34 , and the overenrichment of MitoQ would increase the probability of disrupting mitochondrial homeostasis.We thus reasoned that the property of RET inhibition to increase Δψ m could be exploited to drive mitochondrial enrichment of MitoQ more effectively in tumor cells beyond the threshold to disrupt a bioenergetics balance (Fig. 6).Our data presented in this report strongly support this notion.
Selpercatinib and RET-specific RNA interference consistently increased Δψ m in CCDC6-RET PTC cells, which supports RET-specificity of the selpercatinib effect.Together with our previous demonstration that vandetanib and cabozantinib can increase Δψ m in MEN2A and MEN2B MTC cells 26 , these data suggest that RET is implicated in regulating mitochondrial activity in thyroid tumor cells.In support, Ret overexpression rescued muscle degeneration of the Pink1 deficient Drosophila model of Parkinson's disease (PD) by restoring the mitochondrial respiratory complex I activity partly through upregulating mRNA expression of its subunit, NDUFV2 55 .Of note, complex I inhibition induced mitochondrial hyperpolarization in HEK293 cells 56 , while a complex I defective mutation caused increased Δψ m in neurons 57 .Similarly, knockout of NDUFS4, another complex I subunit, also increased Δψ m in mouse embryonic fibroblasts 58 , although NDUFS4 knockout caused mitochondrial depolarization in mouse skeletal muscle cells 59 .These contrasting effects suggest that complex I activity can conditionally affect Δψ m , probably in a cell context-dependent manner as well as in a temporal-and magnitude-dependent manner.Indeed, short-term inhibition of the complex I by rotenone increased Δψ m whereas its prolonged inhibition by the drug decreased Δψ m in 143B cells 60 .Given this information, it remains to be answered whether different selpercatinib doses can induce differential effects on complex I activity.
By what mechanism does mitochondrial overenrichment of MitoQ induce tumor cell death?MitoQ is expected to function primarily as an antioxidant in cells.Tumor cells depend on reactive oxygen species (ROS) more than normal cells to facilitate tumor cell survival, angiogenesis, metastasis, and chemoresistance 61,62 .In this context, MitoQ would limit tumor cell survival by depleting ROS.Indeed, an oxidized form of ubiquinone (BPM31510) induces anti-cancer effects and is currently tested for Fig. 2 | Selpercatinib synergizes with MitoQ to suppress the viability of RETmutated thyroid tumor cells.a TPC1 cells in 12 well plates were treated with increasing doses of MitoQ, CoQ10, and TPP for 48 hours prior to crystal violet viability assay.Data are expressed as the percentage of untreated controls.Data are mean ± SEM (N = 3).b TPC1 cells, pretreated with different concentrations of selpercatinib, were treated with different doses of MitoQ for 48 hours prior to crystal violet viability assay.Data are mean ± SEM (N = 4).c SynergyFinder-generated plot of the viability data in b.The ZIP (zero interaction potency) score is indicated for the most synergistic area.d TPC1 cells, infected with lentiviral pLKO.1-shRET#1 for 24 hours, were treated with MitoQ for 48 hours prior to crystal violet viability assay.Data are expressed as the percentage of percentage of untreated controls.Data are mean ± SEM (N = 3).e Chou-Talalay plot of the data in d.CI was determined as a function of effect level (Fa).f Western blotting analysis of TPC1 cells cultured under hypoxia.β-actin is the control for equal protein loading.g TPC1 cells pretreated with selpercatinib were treated with MitoQ for 48 hours under the hypoxic condition prior to crystal violet viability assay.Data are mean ± SEM (N = 3).h SynergyFinder plot of the viability data in g.The ZIP score is indicated for the most synergistic area.i TT and MZ-CRC-1 cells pretreated with selpercatinib were treated with MitoQ for 48 hours prior to crystal violet viability assay.Data are mean ± SEM (N = 4).j SynergyFinder plot of the viability data in i.The ZIP score is indicated for the most synergistic area.All data are mean ± SEM (N ≥ 3).*P < 0.05, **P < 0.005, ***P < 0.001, Two-Way ANOVA with Bonferroni post-tests.solid tumors with high ROS burden in clinical trials 63 .An alternative scenario is also available.Although initially designed as an antioxidant, MitoQ can elevate ROS production in cells.Studies using mitochondria isolated from bovine aortic endothelial cells showed that MitoQ can increase ROS generation by mitochondrial complex I while mildly decreasing ROS generation by mitochondrial complex II without any effect on complex III 64,65 .
In contrast, its functional moiety CoQ10 had no similar effects 64,65 .Mechanistically, MitoQ is poorly reduced by complex I because of the bulkiness of TPP moiety that sterically hinders the access of the ubiquinone group to the complex I 66,67 .However, TPP does not affect the access of ubiquinone moiety to the complex II, which makes MitoQ a good substrate for the complex II 67 .Meanwhile, MitoQ is not oxidized by Complex III 66 because the positive charge of the TPP moiety limits the access of MitoQ to the active site(s) of complex III 67 .Of note, MitoQ treatment of endothelial cells increased superoxide production from malate-and glutamate-fueled complex I, leading to the onset of apoptosis 64 .Because many tumor types maintain a higher level of malate and glutamate level to fuel complex I 68 , MitoQ might have pro-oxidant effects in most metabolically active tumor cells.One may question that MitoQ sensitivity of certain normal cell types would limit the use of high dose MitoQ for cancer therapy and requires a strategy to selectively increase MitoQ delivery into tumor cells for effective treatment.We posit that selpercatinib preferentially sensitize RET-mutated tumor cells to MitoQ, proposing that our strategy to combine selpercatinib and MitoQ might confer an advantage in therapeutically using the agent.We also appreciate the possibility that selpercatinib and MitoQ synergize through additional mechanisms.In a tubular injury mouse model and human kidney 2 (HK-2) cells, MitoQ partially attenuated mitophagic cell death caused by PINK/Parkin-deficiency by increasing Nrf2 level 69 .Similarly, MitoQ enhanced PINK/Parkin-mediated mitophagy to inhibit hepatic stellate cell activation and liver fibrosis 70   myocardial ischemia-reperfusion injury in type 2 diabetic rats 71 .Meanwhile, Ret overexpression rescued muscle degeneration of the Pink1 deficient Drosophila PD model 55 , and functional redundancy between RET and Parkin in maintaining mitochondrial function and morphology has been demonstrated in a mouse PD model 72 .Given this information, MitoQ and RET might have effects that overlap in association with PINK.Therefore, it is also possible that RET inhibition and MitoQ may synergistically target a PINK/Parkin-mediated mechanism.Lastly, MitoQ may support the general health status of patients undergoing chemotherapy, as previously suggested 73,74 .Indeed, MitoQ improved muscle atrophy and weakness of the mice bearing colon cancer cell line xenografts 74 .
Clinically, the recommended dosing of selpercatinib for a body weight of ≥50 kg is 160 mg BID, while 120 mg BID for patients weighing <50 kg 23 .The adverse effects of selpercatinib result in permanent discontinuation or dose reduction in about 5% and 31% of patient, respectively 23 .The two patients in this report did not tolerate standard doses of selpercatinib due to adverse effects.However, these patients did tolerate reduced doses of selpercatinib when combined with MitoQ.Whether MitoQ significantly enhanced the effects of selpercatinib cannot be determined in this brief analysis of only two patients.Nevertheless, these observations suggest a strategy to effectively use selpercatinib in the patients with low tolerance to this drug.MitoQ has been confirmed for its safety in humans, and its clinical benefits are currently being evaluated in Phase II trials [32][33][34] , although its benefit for cancer patients has not yet been tested in the setting of a clinical trial.We believe our preclinical data and patient cases are pertinent to the design of a future clinical trial to determine whether selpercatinib and MitoQ combination can provide a clinical benefit to patients with RETmutated cancers.

Cell viability and cell cycle analyses
IC 50 values and combination effects of drug treatments were determined by Crystal Violet (Fisher Chemical, Thermo Fisher Scientific, #C58125) cell viability staining assay.Briefly, cells in 96 well-plates were fixed in formaldehyde (Fisher Chemical, #BP531), stained with 0.05% Crystal Violet for 30 minutes, washed with water three times, air-dried, and incubated in 200 µl of methanol (VWR Chemicals BDH ® , Radnor, PA #BDH1135) for 10 min at room temperature before measuring absorbance at 540 nm.Cell viability for RET knockdown was determined by flow cytometry of cells stained with the fluorescent DNA intercalator Thiazole Red (TO-PRO ® 3, Invitrogen, # T3605) using Guava EasyCyte flow cytometry system (Milli-poreSigma, #05005007).Data were analyzed by FCS EXPRESS software (De Novo Software, Los Angeles, California).For the cell cycle, flow cytometry of cells stained with propidium iodide (PI, Invitrogen, #P1304MP) was performed using the Guava EasyCyte flow cytometry system, as previously described 75 .
Detection of Δψ m using TMRM Cells were incubated with culture medium containing 2 nM TMRM (Invitrogen, #T668) in 12-well plates for 30 min at 37 °C in dark, collected by trypsinization, resuspended in phosphate-buffered saline containing 0.5% bovine serum albumin, and analyzed using Guava EasyCyte flow cytometry system Data were analyzed by FCS EXPRESS software flow, as described previously 27 .

Fig. 1 |
Fig. 1 | Selpercatinib alters mitochondrial membrane potential in cells expressing RET fusion.a TPC1 cells, treated with selpercatinib (selp) in a 3.125 to 50 nM dose range for 2 days, were stained with TMRM.Cellular TMRM retention was analyzed by flow cytometry measuring yellow fluorescence.b Mean fluorescence intensities (MFI) of TMRM-stained cells in a quantified by FCS Express software.Data are mean ± SEM (N = 4).***P < 0.001 (compared with no treatment), one-way ANOVA with Bonferroni post-tests.c TPC1 cells, infected with lentiviral pLKO.1-shRET#1 and shRET#2 for 2 days, were stained with TMRM.Cellular TMRM retention was analyzed by flow cytometry measuring yellow fluorescence.d MFI of TMRM-stained cells in c quantified by FCS Express software.Data are mean ± SEM (N = 3).*P < 0.05 (compared with pLKO.1),one-way ANOVA with Bonferroni post-tests.e PCCL3 cells stably expressing a doxycycline (Dox)-inducible NCOA4-RET were treated with different doses of selpercatinib for 48 hours in the presence or absence of 0.5 µg/mL doxycycline.MFI of TMRM-stained cells was quantified by FCS Express software.Data are mean ± SEM (N = 4).**P < 0.005, ***P < 0.001 (compared with no doxycycline), two-way ANOVA with Bonferroni post-tests.f Western blot analysis of total lysates of PCCL3 cells with or without 48-hour doxycycline treatment.g TPC1 cells pretreated with selpercatinib for 2 days were treated with 2 μM MitoCP for 1 hour.Mitochondrial lysates of these cells were analyzed by western blotting to detect the formation of TPP-adducts using an antibody specific to the TPP moiety of MitoCP.Total proteins were visualized in the stain-free gel as the control for equal protein loading.h Densitometry of the Western blot signals collected from three independent experiments performed as described in g.Signals were normalized for total protein intensity.Data are mean ± SEM (N = 3).**P < 0.005 (compared with no treatment), one-way ANOVA with Bonferroni posttests.i Western blotting to detect TPP-adducts in the mitochondrial lysates of TPC1 cells, infected with pLKO.1-shRET#1 and shRET#2 for 2 days, and then treated with 2 μM MitoCP for 1 hour.Total proteins visualized in the stain-free gel are the control for equal protein loading.j Densitometry of the Western blot signals collected from three independent experiments performed as described in i.Data are mean ± SEM (N = 3).*P < 0.05 (compared with pLKO.1),one-way ANOVA with Bonferroni post-tests.

Fig. 3 |
Fig. 3 | Selpercatinib and MitoQ combination effectively suppresses TPC1 xenografts in mice.a Treatment schedule for a cycle in total four cycles.Detailed information is in Materials and Method.b Changes in tumor sizes at the indicated time points (N = 5).*P < 0.05, **P < 0.005, ***P < 0.001 (relative to the vehicle); # P < 0.05 (combination therapy versus selpercatinib monotherapy).Two-way

Fig. 4 |
Fig. 4 | Treatment of the patient with CCDC6-RET fusion metastatic PTC. a Treatment timeline.b Blood pressure changes during the treatment.Dotted line indicates normal range of blood pressure.c CT scans of lung metastasis (orange arrows) prior to lenvatinib treatment (left), after stopping lenvatinib (middle), and after receiving 7 weeks of the selpercatinib and MitoQ combination therapy (right).Colors in b indicate the same treatment schedule shown in a. d Images of chest wall skin metastasis prior to lenvatinib treatment (left), after stopping lenvatinib (middle), and after receiving 3 weeks of the combination therapy (right).

Fig. 5 |
Fig. 5 | Treatment of the patient with locally advanced, unresectable RET M918T MTC. a Treatment timeline.b Changes in ALT and AST.c Changes in serum calcitonin levels.Dotted lines in b, c indicate normal range of tests; colors indicate the same treatment schedule shown in a. d CT scans of right 7 cm MTC with mass effects on the trachea and esophagus (orange circle) prior to initiating selpercatinib treatment (left), prior to resuming reduced dosage of selpercatinib (middle), and after 7 weeks of the selpercatinib and MitoQ combination therapy (right).

Fig. 6 |
Fig. 6 | Graphic summary of the combination therapy concept.An increase in Δψ m facilitates the mitochondrial accumulation of TPP cations.Because RET inhibition increases Δψ m , a RET inhibitor can enhance mitochondrial enrichment of a TPPconjugated agent beyond a level to disrupt mitochondrial homeostasis and induce cell death.The image was generated with BioRender.com.